The aim of this proposal is to extend our recent findings that i) immature calf and adult steer chondrocytes synthesize different relative amounts of several subpopulations of proteoglycans (PGs) and ii) individual subpopulations exhibit age-related differences in glycosylation. We propose to further elucidate the basis of age-related differences in a) the structure of cartilage (PGs) and b) the metabolism of articular chondrocytes. To further probe age-related changes at the level of biosynthesis, we will determine the structuref and composition of purified subpopulations of aggregating PGs synthesized by phenotypically stable chondrocytes isolated from fetal, immature calf, 18 month old steer and 3-5 year old steer articular cartilage. To determine at which level heterogeneity first arises, we will compare at all ages the intracellular core protein precursors to the core proteins purified from a cell-free translate of RNA and will follow the conversion of the intracellular core protein precursors into the individual subpopulations of PGs. A separate study will establish if the age-related increase in the number of keratan sulfate (KS) chains in chondroitin sulfate-rich PGs occurs at the expense of one or more of the O-linked oligosaccharides which have similar structures. Long term cultures of chondrocytes in agarose gels, which promote phenotypic stability, will be used to study inherent age-related differences in the biosynthesis and turnover of the subpopulations of aggregating as well as non-aggregating PGs laid down within the gel-matrix. Additional studies will probe whether the age-related changes reflect i) changes in the proportion of cells synthesizing different PGs or ii) alterations in the quality of PGs made by all cells. Because of new evidence that serum KS is a marker of cartilage PG catabolism, we will study the elimination of KS-bearing molecules of specific sizes from the circulation. Finally, we propose to further probe the biochemical basis of age-related changes in the expression of the small non-aggregating cartilage PGs.